Limited effects of CTLA4-CD28 gene modification on Pmel-1 T cells in vitro and in vivo. Empty vector–encoding (EV) or CTLA4-CD28 chimera–encoding (CTC28) retrovirus was transduced into activated Pmel-1 T cells with antigenic peptide, human gp100_25-33 (KVPRNQDWL, 1μM). After 2 days of resting in the absence of antigen, GFP-positive T cells were purified by cell sorting. (A) GFP-positive T cells were stimulated with human gp100_25-33 (1μM) in the presence of irradiated splenocytes for 48 hours, and the IFN-γ in the culture supernatant was measured by ELISA. (B) Forty-eight hour–stimulated GFP-positive T cells were cocultured with B16 melanoma cells for 24 hours. Cytotoxicity of the various ratios of T cells to B16 cells (killer/target ratio) were determined by P-JAM test. (C) GFP-positive T cells were expanded in the presence of IL-15 (10 ng/mL) and paramagnetic beads coated with anti-CD3 plus anti-CD28 for 7 days before adoptive transfer to the mice. B6 mice subcutaneously injected with B16 cells were lymphodepleted by total body irradiation (4 Gy) 7 days later, and then they were injected with expanded T cells (2 × 10⁶) intravenously. Thereafter, recombinant human IL-2 (3 × 10⁵ IU/head) was administered intraperitoneally twice a day for 3 days. Mean tumor size of at least 5 mice per group was recorded. Results are representative of 3 independent experiments.